In animal cells, a number of transport processes mediate the passage of purine and pyrimidine nucleosides across the cell membrane (Gati and Paterson (1989), in "Red Blood Cell Membranes", Eds. Agre & Parker, pp. 635-661).
A major nucleoside transport activity in animal cells, including mammalian cells, is the equilibrative (facilitated diffusion) process mediated by a transporter of the plasma membrane which is highly sensitive to inhibition by derivatives of 9-.beta.-D-ribofuranosyl-6-thiopurine such as nitrobenzyl thioinosine (NBMPR). The equilibrative, NBMPR-sensitive nucleoside transporter is referred to herein as the es nucleoside transporter.
The abundance of es nucleoside transporter sites is of significance in predicting the responsiveness of certain leukemias to nucleoside anti-metabolite therapy.
Analysis of equilibrium binding of .sup.3 H-NBMPR has been used to measure the abundance of nucleoside transporter sites in homogeneous populations of a variety of fresh and cultured human cells (Wiley et al. (1987) Aus. & N.Z. J. Med., vol. 17, pp. 379-386; Wiley et al. (1989), Br. J. Haematol. vol. 71, pp. 203-207).). Use of this tritiated ligand does not, however, permit identification of subpopulations of cells with different transporter densities in a heterogeneous cell population, such as usually occurs in human cancer. There are also serious practical limitations to clinical application of NBMPR equilibrium binding to estimating es nucleoside transporter abundance as the method is complex and time-consuming and requires large numbers of cells.
[.sup.125 I]Iodohydroxynitrobenzylthioinosine has been synthesised and used to label the es nucleoside transporter (Gati, Knaus, Wiebe, & Peterson (1987), Biochem. Cell. Biol. vol. 65, pp. 467-473). This iodinated nucleoside is not suitable for use in a clinical application because of high non-specific adsorption of the iodine-labelled nucleoside, the pH-dependence of its binding and the complexity of the assay procedure.
A fluorescent dansyl derivative of 6-thioquanosine has been synthesised and shown to bind to the es nucleoside transporter of human erythrocytes (Shohami & Koren, (1979), Biochem. J., vol. 178, pp. 271-277). The observed high non-specific binding of this derivative make it not as effective as desirable for the basis of a clinically useful technique. Dansyl derivatives also require excitation in the ultraviolet range (310 nm) and are poorly suited to use in flow cytometry techniques.